Keratinocyte stem cells are critical in the processes of tissue homeostasis, wound healing, and carcinogenesis. Nevertheless, mechanisms that underlie these phenomena are largely unknown, mainly due to the lack of molecular markers that could enable separation of stem cells from transient amplifying cells. We have succeeded in isolating presumptive human keratinocyte stem cells and transient amplifying cells using flow cytometry sorting technique. The long term objective of this study is to characterize regulatory mechanisms responsible for epidermal stem cell proliferation and differentiation. The goal of this proposal is to perform a comparative gene expression study of presumptive stem cells and transient amplifying cells. We set forth a plan to systematically analyze different methods for cell handling prior to immunostaning, and then select the optimal procedure for preparing samples. Immunolabeled keratinocytes will be subsequently used for flow cytometry sorting and RNA isolation. Employing genomic tools gene expression profiles of presumptive stem cells and transient amplifying cells will be analyzed. For identification of genes that are uniquely expressed in presumptive stem cells and/or transient amplifying cells, we will use representational difference analysis. This method will enable identification of potential novel genes that are expressed in presumptive stem cells or in transient amplifying cells. Microarray technology will be employed to analyze a large number of genes and identify those that are downregulated, or upregulated in presumptive stem cells, and/or in transient amplifying cells. Obtained results will allow comparisons with existing gene expression data in other stem cell systems and enable evaluation of markers used to identify and isolate presumptive keratinocyte stem cells. These studies are essential for understanding mechanisms of proliferation, differentiation, and maintenance of the stem-like state. Identification of factors involved in these processes is crucial for delineating skin diseases and engineering new therapeutics.